Two cDNA Clones Encoding Isoforms of the B Subunit of the Vacuolar ATPase from Barley Roots

The vacuolar H+-ATPase of higher plants is a member of the V-ATPase family, which comprises complex, multisubunit ATPases found in a11 eukaryotes. The electrochemical gradient created by the V-ATPase is thought to provide the driving force for the iecondary transport of other ions and metabolites (Taiz, 1992). In barley (Hordeum vulgare L.) roots this enzyme may be involved in the sequestration of Na+ and Ca2+ ions in the vacuole, because the proton gradient produced by the ATPase is used by Na+/H+ and Ca2+/H+ antiports to drive the uptake of Na+ and of Ca2+ (Garbarino and DuPont, 1989; DuPont et ai., 1990). The quatemary structure of the ATPase from barley roots is very similar to that from other organisms, with approximately 10 different subunits (DuPont and Momssey, 1992). Two of the best-characterized subunits of the V-ATPases are the A and B subunits of the large, knob-like head group on the cytoplasmic side of the vacuolar membrane. The A subunit is between 67 and 70 kD, and the B subunit is between 53 and 60 kD in various organisms. Both A and B subunits are present in a stoichiometry of three per enzyme complex and contain ATP binding sites, although the B subunit is not directly involved in ATP hydrolysis (Puopolo et al., 1992). The primary structures of the A and B subunits are highly conserved among widely divergent organisms, making them useful for evolutionary studies. Isoforms of the B subunit have been described for mammals, and the distribution of the isofonns seems to be tissue specific (Puopolo et al., 1992). It was of some interest to leam whether there are also isoforms of the B subunit in plants. Two different cDNA clones for the B subunit of the barley V-ATPase, designated HTBl and HTB2, were selected from a barley root cDNA library. The two clones were very similar to each other, having higher sequence identity to each other than to any other B subunit sequence in the GenBank data base (see Table I for details). Since barley is an inbred diploid organism, it is likely that HTBl and HTB2 are encoded by different genes, rather than alleles of the same gene. The next most similar sequence was that for the Arubidopsis